Establishment and characterization of a noradrenergic adrenal chromaffin cell line, tsAM5NE, immortalized with the temperature-sensitive SV40 T-antigen

Abstract

We established a clonal adrenal medullary cell line, named tsAM5NE, from transgenic mice harbouring the temperature-sensitive Simian virus 40 large T-antigen gene, under the control of the tyrosine hydroxylase promoter. tsAM5NE cells conditionally grew at a permissive temperature of 33°C and exhibited the noradrenergic chromaffin cell phenotype. To understand the characteristics of tsAM5NE cells, we first examined the responsiveness of the cells to ligands of the GDNF (glial cell line-derived neurotrophic factor) family. tsAM5NE cells proliferated at the permissive temperature of 33°C in response to either GDNF or neurturin, but not artemin or persephin. At the non-permissive temperature of 39°C, GDNF or neurturin caused tsAM5NE cells to differentiate into neuron-like cells; however, the differentiated cells died in a time-dependent manner. Interestingly, LIF (leukaemia inhibitory factor) did not affect the GDNF-mediated cell proliferation at 33°C, but promoted the survival and differentiation of GDNF-treated cells at 39°C. In the presence of GDNF plus LIF, the morphological change induced by the temperature shift was associated with up-regulated expression of neuronal markers, indicating that the cells had indeed undergone neuronal differentiation. Thus, we demonstrated that tsAM5NE cells had the capacity to terminally differentiate into neuron-like cells in response to GDNF plus LIF when the oncogene was inactivated by the temperature shift. Thus, this cell line provides a useful model system for studying the mechanisms regulating neuronal differentiation.

Methyl galbanate, a novel inhibitor of nitric oxide production in mouse macrophage RAW264.7 cells

“…The RAW264.7 mouse macrophage cell line was obtained from ATCC (Manassas, VA, USA). tsAM5NE, a temperature-sensitive mouse adrenal medullary chromaffin cell line, was used as described [12]. The 6 terpenoid coumarins used in this study were isolated and purified according to the method described previously [4].…”

“…tsAM5NE cells are immortalized mouse adrenal chromaffin cells, in which the temperature-sensitive SV40T antigen gene has been introduced. The cells exhibit norepinephrinergic phenotype, and respond to GDNF and LIF by differentiating into neuron-like cells [12]. RAW264.7 cells were first plated on culture inserts and pretreated with LPS (100 ng/ml)/IFN-c (20 ng/ml) in the presence or absence of methyl galbanate (10 lM) for 6 h in 0.5% FBS-containing medium.…”

Immortality of cell lines: challenges and advantages of establishment

“…When immortality is established, cells do not keep their exact phenotype. Studying this changed phenotypic behaviour may be a key to establishing immortality in its ideal form Kohno et al (2011) Epigenetic changes The immortality of cell lines is affected by epigenetic modifications during cellular differentiation. Simboeck et al (2011) described the epigenetic contribution in the establishment of immortality when studying the alterations in chromatin architecture affecting phenotypic and the epigenetic changes.…”

2.1 Generation of Cell Lines and Biotechnological Applications

“…Growth-controlled cell lines have been established by retroviral transfer of tsTAg into primary cells (e.g., [53]) and also by isolation of cells from a tsTAg transgenic mouse, the so-called Immortomouse ® [54,55]. Examples for tsTAg-based conditionally immortalized cell types encompass myogenic cell lines [56], hepatocyte cell lines [57,58], tissue specific microvascular endothelial cells [59], an astrocyte cell line [60], and more recently an adrenal medullary cell line [61] as well as stroma cell lines [62]. For a more comprehensive overview please see review by Obinata (1997) [63].…”